Adeno-Associated Virus Identity Confirmation in Biopharma by LC/MS

Significance of the Topic

Adeno associated viruses AAVs are widely employed in gene therapy and require rigorous analytical approaches to confirm capsid identity and protein composition. Traditional assays such as SDS PAGE and ELISA often lack resolution and depend on serotype specific antibodies, leading to complexity and potential errors. LC MS presents a rapid, specific and precise alternative, aligning with regulatory expectations for biologics quality assessment.

Objectives and Article Overview

This article presents optimized LC MS workflows for AAV identity confirmation through both intact capsid protein analysis and peptide mapping. It reviews challenges in resolving VP1 VP2 and VP3 capsid proteins, proposes chromatographic strategies, and describes instrumentation and software for accurate mass measurement and post translational modification mapping. Practical recommendations aim to support biopharma laboratories in method implementation and regulatory compliance.

Methodology

Sample preparation involves denaturation and buffer exchange to remove non volatile salts and stabilizers. Intact capsid analysis employs UHPLC separation on wide pore reversed phase columns under high pressure and elevated temperature. Peptide mapping follows protein reduction, alkylation, enzymatic digestion and separation on superficially porous columns. Both workflows integrate a high resolution Q TOF mass spectrometer for mass measurement and sequence confirmation.

Instrumental Setup

Ultralow dispersion LC system equipped with wide pore ZORBAX RRHD columns Diphenyl or StableBond C18 300 Å 1.8 µm 2.1 x 100 or 150 mm or AdvanceBio Peptide Mapping column 2.1 x 150 mm 2.7 µm. Agilent 1290 Infinity II UHPLC with adjustable flow ramps and pressure limits. Agilent 6545XT AdvanceBio LC Q TOF for high mass accuracy. MassHunter BioConfirm software for data analysis and PTM identification.

Main Results and Discussion

Optimized ZORBAX RRHD columns demonstrated clear baseline separation of VP1 VP2 and VP3 capsid proteins, enabling accurate mass determination despite high sequence homology. Flow ramp adjustments and dead volume minimization improved column lifetime and resolution. Peptide mapping on AdvanceBio columns achieved high peak capacity and consistent PTM detection across all capsid proteins. The workflows delivered precise relative abundance measurements and comprehensive sequence coverage, addressing challenges in multi serotype analysis.

Benefits and Practical Applications

The LC MS based methods reduce reliance on labor intensive antibody assays, streamline identity confirmation and enhance specificity for closely related capsid variants. High resolution separations and mass accuracy support regulatory requirements for product release and comparability studies. Buffer exchange and system maintenance guidelines ensure consistent spectral quality and instrument uptime, beneficial for routine QC and research labs.

Future Trends and Potential Applications

As regulatory guidance for gene therapies evolves, peptide mapping may become mandatory for AAV products. Advances in chromatographic materials and high throughput UHPLC platforms will further improve resolution and speed. Integration with automation and cloud based data analysis could enable real time monitoring of manufacturing processes. Emerging MS technologies with higher sensitivity and faster duty cycles will expand capabilities for low abundance variant detection and multi dimensional characterization.

Conclusion

LC MS workflows for intact capsid and peptide mapping of AAVs provide a robust, accurate and efficient solution for identity confirmation and quality assessment in biopharma. The described methods address critical challenges in protein resolution, regulatory compliance and operational practicality, offering a clear path to improved gene therapy analytics.

References

• Backovic A et al Capsid Protein Expression and Adeno associated Virus like Particles Assembly in Saccharomyces Cerevisiae Microb Cell Fact 2012 11 124
• Chemistry Manufacturing and Control Information for Human Gene Therapy Investigational New Drug Applications US Food and Drug Administration Guidance for Industry 2020
• Kuck D Kern A Kleinschmidt JA Development of AAV Serotype Specific ELISAs Using Novel Monoclonal Antibodies Journal of Virological Methods 2007 140 17 24
• Agilent 1290 Infinity II Ultra Low Dispersion Kit Technical Note
• LC MS of Intact Adeno Associated Virus AAV Capsid Proteins for Product Identity Application Note 5994 2434EN
• Characterization of Viral Vector Particles Using the Agilent 6545XT AdvanceBio LC Q TOF Application Note 5994 1980EN